Kinetic Assembly of Bispecific Antibodies Bispecific antibodies (BiAb) are a promising class of protein pharmaceuticals utilized to target and/or redirect therapeutic cells as well as overcome regulatory hurdles for therapies affecting two targets. Numerous BiAbs are under development to direct activated T cells to tumors. Our laboratory (Lee, 2006) is utilizing this approach to address a major problem in stem cell (SC) therapy: targeting to and retention of SC at sites of tissue injury, e.g. myocardial infarction, stroke, macular degeneration, etc. Production of BiAbs by hybrid hybridomas or chemical crosslinking suffers from several disadvantages, such as aggregation and possible inactivation (in the case of chemical ligation), inefficiency of production and separation of unwanted byproducts. Attempts to solve these problems by genetic engineering have been largely unsuccessful, raising new problems, including reproducibility and potency. No currently available routine techniques permit commercial-scale production of bispecific antibodies. To address this need, we have designed a novel strategy to create KinAbs(tm), kinetically assembled BiAbs, that will result in efficient BiAb production. KinAbs(tm) employ the foot and mouth disease virus 2A (FMDV-2A) self-processing cleavage sequence to force association of two heavy chains by expressing them from one ORF so that they can begin to associate while still being translated on ribosomes, in effect borrowing the same mechanism used by FMDV to assemble and process polyproteins. Fang et al. (2005) found this mechanism to be highly effective in forcing association of light and heavy chains to more efficiently produce Abs. Unlike most other methods, KinAb(tm) technology can be used to produce structurally normal Abs that possess two specificities. However, in order to facilitate homogeneous BiAb production, we also have created a new scFv-Fc format (LA-BiAb") in which one scFv-Fc uses the kappa-2 light chain variant that cannot bind protein L, and the other scFv-Fc carries a mutant Fc that cannot bind protein A. We utilize sequential purification with protein L and protein A affinity chromatography to isolate pure BiAbs. KinAbs(tm) and LA-BiAb(tm) are independent technologies that can be combined for generalizable, efficient, simple production of BiAbs to create a new generation of bispecific therapeutics and diagnostics. [unreadable] PUBLIC HEALTH RELEVANCE: Kinetic Assembly of Bispecific Antibodies Bispecific antibodies (BiAb) are a promising class of protein pharmaceuticals utilized to target and/or redirect therapeutic cells as well as overcome regulatory hurdles for therapies affecting two targets. Numerous BiAbs are under development to direct activated T cells to tumors. Our laboratory (Lee, 2006) is utilizing this approach to address a major problem in stem cell (SC) therapy: targeting to and retention of SC at sites of tissue injury, e.g. myocardial infarction, stroke, macular degeneration, etc. Production of BiAbs by hybrid hybridomas or chemical crosslinking suffers from several disadvantages, such as aggregation and possible inactivation (in the case of chemical ligation), inefficiency of production and separation of unwanted byproducts. Attempts to solve these problems by genetic engineering have been largely unsuccessful, raising new problems, including reproducibility and potency. No currently available routine techniques permit commercial-scale production of bispecific antibodies. To address this need, we have designed a novel strategy to create KinAbs(tm), kinetically assembled BiAbs, that will result in efficient BiAb production. KinAbs(tm) employ the foot and mouth disease virus 2A (FMDV-2A) self-processing cleavage sequence to force association of two heavy chains by expressing them from one ORF so that they can begin to associate while still being translated on ribosomes, in effect borrowing the same mechanism used by FMDV to assemble and process polyproteins. Fang et al. (2005) found this mechanism to be highly effective in forcing association of light and heavy chains to more efficiently produce Abs. Unlike most other methods, KinAb(tm) technology can be used to produce structurally normal Abs that possess two specificities. However, in order to facilitate homogeneous BiAb production, we also have created a new scFv-Fc format (LA-BiAb") in which one scFv-Fc uses the kappa-2 light chain variant that cannot bind protein L, and the other scFv-Fc carries a mutant Fc that cannot bind protein A. We utilize sequential purification with protein L and protein A affinity chromatography to isolate pure BiAbs. KinAbs(tm) and LA-BiAb(tm) are independent technologies that can be combined for generalizable, efficient, simple production of BiAbs to create a new generation of bispecific therapeutics and diagnostics. [unreadable] [unreadable]